This invention relates to a novel amino acid derivative, a method for preparing the same and a method for preparing human osteocalcin using the same which is an important index of bone metabolism and aging of human being.
Heretofore, as .gamma.-carboxyglutamic acid derivatives as a starting material for chemical synthesis of partial fragment of a protein and a peptide, the following has been known. ##STR2## wherein X, R and R' each represent a combination as shown in the following table.
______________________________________ Compound X R R' ______________________________________ a Cbz CH.sub.3 tBu b Cbz Bzl tBu c Cbz CH.sub.3 Bzl d tBoc CH.sub.3 Bzl e Cbz Phac tBu ______________________________________
wherein Cbz represents a benzyloxycarbonyl group, tBu represents a t-butyl group, Bzl represents a benzyl group, tBoc represents a t-butyloxycarbonyl group and Phac represents a phenacyl group.
However, when the above compound is used as a starting material for the compounds shown in the above table, using the benzyloxycarbonyl (Cbzl) group as the .alpha.-amino protective group X is possible only in the case of the liquid phase method and it is not used or cannot be used (Compounds a, b, c and e) in the solid phase method or partial solid phase method. Also, regarding the compound d, when it is used in the solid phase method and in the partial solid phase method, a problem which exist is that selectively eliminating of only the .alpha.-carboxyl protective group R while leaving the protective groups R intact and R' is impossible.
The present inventors have found that in the above compound represented by the formula, a compound represented by the formula (I) mentioned below can be selectively deprotected for the above groups X, R and R' can be utilized in the solid phase method can be the partial solid phase method.
Osteocalcin (Bone Gla Protein: BGP) is a vitamin K dependent calcium bindable protein comprising 15 to 20% of the non-collagenic proteins of bone, and has been considered to be intimately related to both bone formation and bone absorption [Journal of Bone Metabolism Society of Japan 4, 56, 1986]. Poser et al analyzed the primary structure of human osteocalcin, and reported that human osteocalcin is a mixture of Glu.sup.7 osteocalcin with the 17-position being glutamic acid and Gla.sup.7 osteocalcin with the 17-position being .gamma.-carboxyglutamic acid having the amino acid sequence shown below existing at a ratio of 91:9 [Poser, J. W. et al., Proc. Natl. Acad. Sci. U.S., 255, 8685-8691 (1980)]. ##STR3## wherein X represents .gamma.-carboxyglutamic acid residue (Gla) or glutamic acid residue (Glu).
However, osteocalcins of cattle, swordfish, cat, chicken, rat, goat, pig and rat already known up to date are all .gamma.-carboxyglutamic acid at the corresponding site. As the reason, Poser et al estimate that calcified bone of aged people is used as the extraction material of human osteocalcin, and glutamic acid at the 17-position will change to .gamma.-carboxyglutamic acid as man is older.
From such standpoint, establishment of human osteocalcin measuring system is not only clinically useful for diagnosis of bone diseases such as Paget's disease, bone metastasis, etc., but also can be expected to examine the relationship with aging by preparing the antibodies and antisera which recognize individually the 17-position Gla and Glu of human osteocalcin.
However, there exist none of measuring systems for quantitating separately Gla.sup.7 human osteocalcin and Glu.sup.7 human osteocalcin as a matter of course, and also human osteocalcin measuring system by use of human osteocalcin as the standard product. This is because Gla.sup.7 human osteocalcin and Glu.sup.7 human osteocalcin for specific antibody or antiserum preparation, and also as the standard product are not available.
In the present specification, the abbreviations, abbreviated symbols employed have the following meanings.